Chronic pain associated with temporomandibular disorders (TMD) and muscles of mastication presents a significant health problem in the US and across the globe. Reactive oxygen species (ROS) [as prooxidants encompassing reactive nitrogen species], which can induce oxidative stress, play a critical role in the pathogenesis of orofacial pain associated with the TMD, but the molecular basis for their role is not known. We have developed a chronic orofacial pain model based on the effects of a prooxidant, which decays readily in vivo to the same free radicals (e.g., ROS) produced by activated phagocytes, which maintain the inflammatory pain process by reducing cellular antioxidant defenses. While no animal model can recapitulate the full spectrum of human TMD symptoms and pathologies, but the proposed model is most relevant to a subset of human TMD involving metabolic (autoimmune) and infectious arthritis. ROS can initiate pain signaling through the Toll-like receptor-4 (TLR4), a transmembrane receptor for innate and adaptive immunity. The ROS/TLR4-coupling will modify the activation pathway of nuclear factor kappa B (NF:B) by increasing the activity of c-Src, a non-receptor tyrosine kinase. Through this pathway, ROS upregulates the expression of P2X3 receptor (P2X3R), a critical ATP-gated channel that is implicated in chronic orofacial pain and which is highly expressed on sensory neurons that innervate the masseter. Thus ROS/TLR4 coupling initiates and maintains pain transmission by muscle afferents and the trigeminal ganglia by reducing cellular antioxidant defenses. We will use this novel chronic orofacial pain model to investigate the mechanism(s) that integrates ROS/TLR4-coupled signaling to P2X3R dysregulation in the masseter afferents and trigeminal ganglia (TG)/brainstem pain pathway. The long-term goal of the proposed studies is to determine the mechanism(s) by which the ROS/TLR4-coupled signaling mediates orofacial pain. We will test the central hypothesis that ROS-induced TLR4 stimulation will increase NF:B activation by c-Src to up-regulate the expression of P2X3R, and promote and maintain pain sensitivity. To test this hypothesis, we propose the following two Specific Aims. In the first Specific Aim, we will characterize a novel and chronic pro-oxidant-based orofacial pain model. In the second Specific Aim, we will determine the mechanistic linkages involved in prooxidant-induced pain pathway through increase in Src/NF:B-coupled activation and P2X3R expression. A major scientific impact of the results of the proposed studies will be to provide a novel insight on the mechanism(s) that regulate functional P2X3R by ROS/TLR4-coupled NF:B activation in orofacial pain. PUBLIC HEALTH RELEVANCE: Chronic orofacial pain represents a significant burden to society. The proposed experiments will develop and characterize a novel model of orofacial pain to provide a fundamental knowledge on the link between oxidant stress and the development of chronic orofacial pain. By identifying the relevant biological pathways, these studies could uncover novel therapeutic targets for chronic orofacial pain management.